PROJECT SUMMARY/ABSTRACT Currently, over 13 million Americans, including teenagers and adults, use electronic nicotine delivery systems (ENDS), which were first introduced to the United States (U.S.) in 2006. In 2019, there was an outbreak of e- cigarette (e-cig) or vaping-associated lung injury (EVALI) across the U.S., with more than 2,700 cases of lung injury plus 60 associated deaths. Clearly, all ENDS are not ?safe?, and more research on vaping health outcomes is urgently needed. The EVALI outbreak was associated predominantly with the presence of vitamin E acetate (Vit EA) in tetrahydrocannabinol (THC)-containing products; Vit EA was found in the broncho-alveolar lavage fluid (BALF) of EVALI patients reporting use of THC products. A causal relationship, however, between Vit EA and EVALI remains unclear, since EVALI has been diagnosed in patients since 2012, and is still observed in nicotine-exclusive ENDS users. In more than 50% of EVALI patients, the BALF contained lipid-laden macrophages, which have been associated with immune-inflammatory responses and unbalanced levels of surfactant that can evolve into lipoid pneumonia or pneumonitis-like reactions, as seen in EVALI. Besides Vit EA, propylene glycol (PG) and vegetable glycerin (VG), the main e-liquid constituents, are biologically relevant molecules with emulsifying properties that could impair lipid homeostasis in the lungs. Our preliminary data demonstrate 1) that Vit EA e-cig aerosol is detrimental to lung epithelial cells in vitro, and 2) that PG and VG e- cig aerosol induce molecular changes associated with concentration of lipid in the lungs of mice. Therefore, we hypothesize that long-term inhalation of e-cig aerosols containing either PG, VG, Vit EA or a combination of those humectants, will induce lipid-mediated lung injury leading to a decline in lung function, both hallmarks of EVALI. The constant evolution of the ENDS industry makes ENDS-related research challenging and complex. Our approach will circumvent current research limitations by investigating the long-term pulmonary effects of inhaling Vit EA, plus PG and VG, the two most widely used and common humectants found in the formulation of e-liquids of ENDS. The overarching goal of this project is to define the roles that PG, VG, Vit EA or a combination of those humectants, as main constituents of e-liquid formulations and associated e-cig aerosols, play in the health outcomes from vaping, using physiologically relevant in vitro and in vivo models. Specifically, we will 1) determine whether PG, VG, Vit EA or a combination of those humectants can affect the inflammation and immune responses of human pulmonary epithelial cells and macrophages; and 2) screen for EVALI biomarkers that can serve as predictive indicators of EVALI progression in vivo in a well-characterized mouse model of inhalation exposures. The expected results could assist in establishment of new regulatory guidelines regarding the use of PG, VG and Vit EA as inhalants from ENDS devices. Additionally, since EVALI can be fatal, a better understanding of EVALI biomarkers could help address an unmet medical need in EVALI management and treatment.